Chaperonin point mutation enhances cadmium endurance in Saccharomyces cerevisiae

To study the effect of the mutation in conserved G412E in Cct7p subunit of CCT complex on its cellular fate. TriC/CCT is a dynamic multimeric protein that assists in protein folding in an energy-dependent manner. A point mutation in the ATP binding pocket in the equatorial domain of the Cct7p subunit delays the doubling time. The cell size was twice the wild type, and the formation of protein aggregates suggests disturbed folding of the proteins. Upon growing in stressful conditions of arsenous acid and cadmium chloride, the mutant was lethal in As3+ but grew well in Cd2+ with 10.5 µg cadmium uptake mg−1 compared to the wild type. The increased expression of vacuole transporters YCF1 and BPT1 by ten-fold and two-fold in mutant indicates the metal transportation to the vacuole. CCT complex was vulnerable to the mutation in G412E in the Cct7p subunit of protein folding molecular machinery. Interestingly, already stressed cells provided robustness against oxidative stress and cadmium sequestration in the vacuole.


Introduction
Chaperones are required for proper folding of the nascent polypeptide synthesis ensue in the lumen of the cell. These chaperones bind non-covalently with the exposed hydrophobic surfaces of newly formed polypeptides to prevent them from misfolding. Molecular chaperones are essentially not an element of the final form of a protein. The chaperonin TriC/CCT complex folds approximately 10% of cellular proteins, including cytoskeleton protein actin and tubulin (Thulasiraman et al. 1999;Yam et al. 2008). The CCT complex is composed of 16 subunits encoded by eight paralogous essential genes CCT1-8. The arrangement of subunits is in a ring-shaped manner containing eight related but different subunits. Two such rings are stacked upon each other to assemble into a cylindrical structure with a central cavity. This cavity provides a secluded environment for native protein to fold into its three-dimensional functional form. Each subunit comprises three domains-apical, intermediate, and equatorial. The apical domain interacts with the substrate to be folded by the complex and contains a helical extension that forms the lid. It has open and close forms that enable the folding of the substrate by binding and hydrolysis of ATP in the equatorial domain. Intermediate domain coordinates between these two domains (Spiess et al. 2004;Gestaut et al. 2019). Each subunit has a distinct affinity to ATP, and hydrolysis occurs sequentially, aiding its diverse substrate folding (Shimon et al. 2008;Gruber et al. 2017).
Loss of function mutation in any of the subunits often disturbs protein folding. Mis-folded proteins have exposed hydrophobic moiety that may form aggregates with other polypeptides present in overly crowded cytosol. In neurodegenerative disorders like Alzheimer's (AD) and Huntington's (HD) diseases, cellular protein aggregation occurs in neurons. A mutation altering aspartate to glutamate in the P-loop of ATP binding pocket in Cct3p leads to loss of interaction with Q/N rich protein aggregates (Carmichael et al. 2000;Nadler-Holly et al. 2012). Oxidative stress and protein aggregates are indicators of neurodegenerative disorders. Oxidative stress causes oxidative peroxidation of unsaturated fatty acids and produces 4-hydroxy-2, 3-neonenal (HNE). In Alzheimer's disease, levels of HNE in plasma are high (Selley et al. 2002). A decline in glutathione levels was seen in Parkinson's disease and determined as a potential biomarker in the disorder's progression (Mischley et al. 2016). Saccharomyces cerevisiae is a well-established eukaryotic model system with a defined genetic system and relatively facile genetic manipulation methods. It has been extensively used to understand many human diseases due to the similarity of core biological processes. For example, the expression of alpha-synuclein A53T mutant induces abnormal folding of HD proteins in yeast models, leads to the discovery of CCTs ability to alleviate the phenotype (Sot et al. 2017).
The GGG (412-414 in Cct7p) motif of ATP binding pocket in the equatorial domain is conserved from bacterial counterpart GroEL to human CCT. Moreover, the motif is conserved amongst the eight subunits of yeast except in Cct6p and Cct8p, in which GAG sequence is present instead of GGG. As the GGG motif occurs in the equatorial domain, it is predicted to have a role in ADP binding (unpublished docking data; Chagoyen et al. 2014). The subunits Cct6p and Cct8p remain ADP-bound and do not participate in the ATPase cycle of the complex (Jin et al. 2019). Mutation in the GGG motif of Cct1p leads to temperature-sensitive phenotype, whereas the same mutation in Cct6p does not affect its growth (Lin et al. 1997;Ursic et al. 1994). Herein, we have created a point mutation by modifying glycine to aspartic acid at 412 position (G412E) of the amino acid sequence in Cct7p to study the role of the conserved motif GGG (412-414) of ATP binding pocket. The mutation caused growth defects, altered cell morphology, and protein accumulation. We have also observed a possible cross-talk between aggregation and oxidative stress pathways.

Vectors, plasmid constructions and primers
The yeast vectors pRS315, pRS316, and pRS11326 were used for maintaining constructs and assessing the phenotypes. The plasmid pUC19 was used for deletion construct. The CCT7 gene (2153 bp) was amplified by PCR using primers KC124 and KC125 cloned into pJET1.2/blunt cloning vector. Subsequently, it was subcloned into pRS316 at BamHI-SalI site generating the plasmid pKA376. Upstream (591 bp) and downstream (506 bp) sequences of CCT7 were amplified using the primers KC126/KC127 and KC128/KC129. The PCR amplified products were cloned into vector pTZ57R/T. The primers KC120 and KC121 were used to amplify 1205 bp TRP1 gene and cloned into pJET1.2/blunt cloning vector. Plasmid pUC19 was used as a primary vector for plasmid constructs. 1.2 kb TRP1 gene was cloned in BamHI-SalI site and 591 bp BamHI-SacI upstream fragment was cloned in the BamHI-SacI site of pUC19. SphI-SalI 506 bp downstream fragment was cloned in the above construct in SphI-SalI site. The cassette was released by digesting with SacI and transformed into yeast strain B-8728 containing a copy of CCT7 in pRS316 plasmid. Gene deletion was done using the homologous gene disruption method. The primers KC316/KC317 were used for internal amplification and KC542/KC129 were used for junction checking to verify the gene deletion. A point mutation was created from GGG to EGG of CCT7 using primers KC474/KC475 with CCT7 cloned in BamHI-SalI site of pRS315 using sitedirected mutagenesis kit. The primers KC589/KC590 were used to amplify the HSP104 gene (3181 bp, promoter and 3' end forming sequences) and cloned in BamHI-XhoI site of pRS11326 for the aggregation visualization. Subsequently, a DNA fragment (750 bp) containing GFP was cloned at BamHI site to generate the plasmid pKA844. The GFP-tagged cassette was transformed in wild-type and mutant yeast (Sambrook and Russell 2001). The lithium acetate/ polyethylene glycol method was used to transform yeast cells (Hinnen et al. 1978). All the strains, plasmid constructs and primers used in the study are listed in Supplementary Tables 1, 2, and 3.

Atomic adsorption spectroscopy
Fresh yeast cells were inoculated in 20 ml of YPD, grown till the early log phase. 1 mM CdCl 2 was added to it and grown for 16 h at 30°C, initial pH 6. Conditions were altered accordingly to study the effect of contact time and pH. The culture was centrifuged at 5000 rpm for 5 min, and the supernatant was analyzed for residual cadmium by Perkin Elmer AAS, PinAAcle 900F. The Q, uptake capacity (lg mg -1 ) and E, removal efficiency (%) was calculated by where Ci (lg ml -1 ) is the initial concentration, Ce (lg ml -1 ) is the final concentration of cadmium, M (mg) is the dry weight of yeast, and V (ml) is the volume (Pérez-Marín et al. 2007).

Microscopic methods
Strains were grown till the early-log phase then 1 mM CdCl 2 was added and grown for 4 h with and without cadmium. Cells were collected and washed with distilled water. Slides were prepared in 1% agar and seen under the Nikon ECLIPSE Ti-Efluorescent microscope. For electron microscopy, cells were spread over 1% agar, layered the coverslip, and desiccated. These were then sputtered with gold particles for 15 s and imaged using scanning electron microscope Hitachi SU6600 (Japan). 5 ll of 2 mg neutral red ml -1 was added to 25 ll of cell culture and incubated for 5 min to visualize vacuoles, and slides were seen under the optical microscope (Corbacho et al. 2010).

RNA preparation and real-time PCR
Total RNA isolation was done using the hot acidphenol method as described. Eppendorf NanoDrop spectrophotometer was used for estimation of RNA concentration. DNaseI treatment was given to remove any DNA contamination in RNA samples and verified with standard PCR. cDNA from RNA samples were prepared using Thermo Fisher Scientific RevertAid first strand cDNA synthesis kit. Real-time quantitative PCR was performed using Bio-Rad CFX96 Real-time system in 20 ll reactions taking 25 ng cDNA ll -1 in each sample using Thermo Fisher Scientific Maxima SYBR Green/ROX qPCR Master Mix. The in-built Bio-Rad system protocol (CFX_2 step ampl protocol) was used for RT-qPCR with additional melt curves after the amplification. Briefly, denaturation was done at 95°C for 3 min, followed by 40 cycles of 95°C for 10 s and 55°C for 30 s. After amplification, melt curves were run at 65-95°C for 5 s at increment of 0.5°C. The gene expression difference was calculated as fold change (2 -DDCT) using the DDC T method (Schmittgen and Livak 2008). DC T is difference between C T of target gene and C T of reference gene and DDC T is difference between DC T of mutant and wild type strain. Triplicates were performed in all the PCR reactions.

Bioinformatics methods and tools
Peptide sequences of eight yeast CCTs were retrieved from the Saccharomyces Genome Database. Prime module (version 11.1) of Schrödinger, (Schrödinger, LLC, New York, NY, 2017) was used to predict threedimensional structures. Template structures 6KRD_8 was selected based on the percentage identity. ATP docking was carried out using swissdock online tool (Grosdidier et al. 2011). Clustal omega was used for multiple sequence alignment of protein sequences. Web logo model for conserved motif was prepared using consurf (Ashkenazy et al. 2016;Sievers et al. 2011).

Results and discussion
CCT subunits have varying affinity to ATP of Cct4p, Cct5p, Cct2p, Cct1p, Cct7p, Cct3p, Cct6p and Cct8p in descending order. These subunits are characterized on the basis of affinity hierarchy into two asymmetrical planes of high and low-affinity subunits. Cct4p, Cct5p, Cct2p, Cct1p have higher affinity than subunits Cct3p, Cct6p, and Cct8p, while the status of Cct7p remains ambiguous (Amit et al. 2010;Reissmann et al. 2012;Zang et al. 2016). The four conserved loop motifs LGPKG, ITKDG, GDGTT, and GGG are identified in equatorial domains of chaperonins (Kabir et al. 2011). These motifs are differentially conserved among subunits of yeast CCTs and attribute to asymmetry to ATPase activity (Chagoyen et al. 2014). The P-loop GDGTT is extensively studied in Fig. 1 Growth assay: a Spot assay of wild type and mutant on YPD plate at dilutions 10 4 , 10 3 , 10 2 , and 10 1 CFU per spot as indicated. b Liquid growth assay of wild type and mutant on YPD c. Modelling of Cct7p depicting location of conserved glycine residue at 412 position of equatorial domain and ConSurf model represents the conserved glycine residue in all eight subunits Fig. 2 Spot assay of wild type and mutant on YPD plate containing 1 mM of arsenous acid, 1 mM of sodium arsenate, 2 mM of copper sulphate, 2 mM of zinc sulphate and 1 mM of cadmium chloride at 30°C at dilutions 10 4 , 10 3 , 10 2 , and 10 1 CFU per spot as shown Fig. 3 Spot assay wild type and mutant on YPD plates containing 1 mM Cadmium chloride at 30°C at pH4, 6 and 7 at dilutions 10 4 , 10 3 , 10 2 , and 10 1 CFU per spot as indicated all eight subunits of yeast CCTs by creating residue modification. High-affinity CCT1 and low-affinity CCT6 on mutating in GGG motif are temperature sensitive and phenotypically unaffected respectively (Lin et al. 1997;Ursic et al. 1994). However, CCT7 remains poorly understood in the context of its binding affinity to ATP. Hence we have made an effort to incorporate the mutation G412E in the conserved GGG region of CCT7 and analyzed it. In haploid Saccharomyces cerevisiae strain B-8728, the CCT7 gene in the genome was deleted by homologous recombination, providing an extra copy of the gene in pRS316 plasmid (CEN6 URA3). The size of the deletion cassette used was 2.2 kb. The accurate deletion was confirmed using two sets of PCR primers to amplify the 5' and 3' junctions ( Supplementary  Fig. 1 a.). One of the two primers used for junction bind in the marker sequence, and the other corresponds to the sequence outside the flanking sequences used for deletion cassette. The primer KC542/KC317 and KC129/KC316 were used to check upstream and downstream junctions, respectively. The expected size for upstream junction PCR product was 921 bp and 850 bp for the downstream junction ( Supplementary  Fig. 1a and 1b). The cct7 deletion mutants were selected on the plate containing 5-fluoroorotic acid (FOA). As CCT7 is an essential gene, deletant mutant will not grow on FOA plate. The wild type copy of CCT7 in pRS316 (CEN6 URA3) was replaced by mutant CCT7 (G412E) present in pRS315 (CEN6 LEU2) by plasmid shuffling.
Effect of mutation on growth CCT mutants have growth defects, as reported in earlier works (Lin et al. 1997;Narayanan et al. 2016). G412E mutant in Cct7p subunit has visible growth retardation in complete media at optimum temperature ( Fig. 1). Doubling time was 3.47 ? / -0.2 h and 2.05 ? / -0.2 h of mutant and wild type, respectively. Actin is obligate TriC/CCT substrate that plays a central role in cytokinesis. Cdh1p and Cdc20p are  (Kaisari et al. 2017;Llorca et al. 2000). Similarly, slow growth was seen in a strain carrying cct7 mutation in the conserved GDGTT motif of ATP binding pocket (Amit et al. 2010). Substrate folding takes place by open and close conformation cycle, which is powered by ATPase activity. Strain with the G345D mutation in the hinge region of cct4p has slower ATP hydrolysis (Shimon et al. 2008). Glycine at 412 position is localized close to P-loop (GDGTT) of the binding pocket; mutation will likely disturb the ATP binding. Docking of individual subunit study shows that wildtype Cct7p binds tightly than mutant cct7p (Supplementary Fig. 2). The yeast strain carrying mutant cct7 was investigated in various stress conditions to assess its sensitivity. Cell growth was at par with optimum conditions at acidic to alkaline pH, oxidizing agent hydrogen peroxide and redox agent dithiothreitol (DTT) however lesser than wild type ( Supplementary  Fig. 3).
Metal ions bind to the thiol group of cysteine residue and affect cellular proteins. Metal ions have a propensity to produce reactive oxygen species that causes protein damage. Mutant viability was tested against different metal salts, namely zinc chloride, copper sulphate, cadmium chloride, arsenous acid, and sodium arsenate. Except for arsenous acid (III), no further growth differences were observed than wildtype strain. On the contrary to As 3? , cells were growing better than wild type in 1 mM cadmium chloride (Fig. 2, Supplementary Fig. 4). As 3? inhibits CCT complex and tubulin, actin polymerization by binding to its monomer (Pan et al. 2010;Thorsen et al. 2009). Cadmium produces protein aggregates in yeast; Heat shock proteins (HSPs) are up-regulated in the presence of both As 3? and Cd 2? (Jacobson et al. 2017). Mutant is growing in Cd (II) better than the wild type; tolerance to the metal ion is attributed to the outflux of metal ion or hyperaccumulation. Cadmium uptake efficiency is 10.5 lg cadmium mg -1 and 9 lg cadmium mg -1 for mutant and wild type, respectively. Cells were grown for 16, 24, and 48 h to study the effect of contact time on cadmium adsorption of mutant. The removal capacity was 42.75%, 46.70%, and 65.17% for 16, 24, and 48 h respectively. Mutant and wild type were grown in pH 4, 6, and 7 with 1 mM CdCl 2 . Mutant can grow better at acidic pH4 with a removal capacity of 48.97% compared to pH6 with 42.75% (Fig. 3). Cadmium removal capacity was favoured by acidic pH4 and 48 h contact time.

Aggregation of the protein
The heat shock protein 104 (Hsp104) is a cytosolic protein that, on the incidence of protein aggregates, gets recruited on-site and stimulates Hsp70 to bind this complex and assists in clearance. To visualize protein aggregates, Hsp104 was tagged with GFP and expressed in both wild type and mutant. In wild type, Hsp104 was dispersed all over the cytosol while  Relative amplification for C-DNA of wild type and mutant was loaded in lane 1, 3, 5 and 2, 4, 6 respectively. Lane 1, 2; 3, 4 and 5, 6 contains reference gene RDN18-1, YCF1 and BPT1 respectively on 2% agarose gel aggregates were present in the mutant. These aggregates may have a protective function against oxidative stress. The amyloid plaque has metal chelation and antioxidant properties at macromolecular metal levels Fischer 2008, 2009;Carija et al. 2017). The aggregate formation was seen in both wild type and mutant when grown in the presence of cadmium (Jacobson et al. 2017) (Fig. 4).

Vacuole sequestration of cadmium and altered cell morphology
The mutant's cell size was enlarged and is roughly double the size of the wild type (Fig. 5). Larger cell size can be due to prominent vacuoles in the cell (Chan and Marshall 2014). For visualization, wild type and mutant were then stained with vacuole dye neutral red (Corbacho et al. 2010), mutants were able to retain more stain compared to wild type (Fig. 6a). It is evident that mutant has more pronounced vacuoles and possibly assisting in withstanding cadmium. Mutant was sensitive to lactic acid. Lactic acid causes vacuole fragmentation (Suzuki et al. 2012) (Fig. 6b). Aggregated proteins are subjected to degradation via proteasome and vacuolar pathways. Cct1-1, Cct1-2, Cct1-3, Cct2-1 and Cct6-50 are also sensitive to lactic acid. (Petroi et al. 2012;Narayanan et al. 2016) Metal chelation and vacuole sequestration are often adopted strategies in metal tolerant strains. Ycf1p and Bpt1p are vacuole ABC transporters (Jacobson et al. 2017;Sharma et al. 2002). These transporters influx metal ions bound complexes of glutathione to the vacuoles. Relative expression of the YCF1 and BPT1 genes was enhanced by ten-fold and two-fold in the mutant, respectively. RDN18-1 was taken as the reference gene (Fig. 7). The mutation leads to enhanced expression of the two genes supported by sensitivity to lactic acid and thus prepared mutants for detoxification of Cd 2? .

Conclusion
The chaperonin CCT complex is a central molecule that, along with translation machinery, HSPs and ubiquitin, tightly maintains cellular protein homeostasis. Loss of proper folding or degradation leads to disturbance in cell physiology, such as in neurodegenerative disorders. The Cct7p subunit of the CCT complex has exhibited sensitivity to G412E mutation. Cells were enlarged, has slow doubling time, and presence of protein aggregates. Surprisingly, the mutant was generally resistant to oxidative stress and accumulated cadmium better than the wild type. An increase in the expression level of metal ion vacuolar transporters genes, YCF1 and BPT1, in mutant suggests metal influx to vacuoles. This study provides a cross-talk between protein folding and stress pathways. Further research is needed to understand how a mutation in CCT prepares the cell for oxidative stress and overexpression of vacuolar transporters.